18f-labelled folates

ABSTRACT

The present invention is directed towards new  18 F-folate radiopharmaceuticals, wherein the fluorine-18 is covalently linked to the aminobenzoyl moiety, which connects the condensed pyrimidine heterocycle to the amino acid portion within folate structures, as well as their precursors and their non-radioactive references, a method of their preparation, as well as their use in diagnosis of a cell or population of cells expressing a folate-receptor and monitoring of cancer and inflammatory and autoimmune diseases and therapy thereof.

FIELD OF INVENTION

The present invention is directed towards new ¹⁸F-folateradiopharmaceuticals, wherein fluorine-18 is covalently linked to theaminobenzoyl moiety, which connects the condensed pyrimidine heterocycleto the amino acid portion within folate structures, as well as theirprecursors, a method of their preparation, as well as their use indiagnosis of a cell or population of cells expressing a folate-receptorand monitoring of cancer and inflammatory and autoimmune diseases andtherapy thereof.

BACKGROUND

Cell-specific targeting for delivery of effector moieties such asdiagnostic or therapeutic agents is a widely researched field and hasled to the development of non-invasive diagnostic and/or therapeuticmedical applications. In particular in the field of nuclear medicineprocedures and treatments, which employ radioactive materials emittingelectromagnetic radiations as 7-rays or particle emitting radiation,selective localization of these radioactive materials in targeted cellsor tissues is required to achieve either high signal intensity forvisualization of specific tissues, assessing a disease and/or monitoringeffects of therapeutic treatments, or high radiation dose, fordelivering adequate doses of ionizing radiation to a specified diseasedsite, without the risk of radiation injury in other e.g. healthytissues. It is thus of crucial interest to determine and assesscell-specific structures and in particular structures that are presentin case of cancer (i.e. tumors) or inflammatory and autoimmune diseases,such as receptors, antigens, haptens and the like which can bespecifically targeted by the respective biological vehicles.

The folate receptor (FR) has been identified as one of these structures.The FR is a high-affinity (K_(D)<10⁻⁹ M) membrane-associated protein. Innormal tissues and organs FR-expression is highly restricted to only afew organs (e.g. kidney, lungs, choroids plexus, and placenta), where itlargely occurs at the luminal surface of epithelial cells and istherefore not supplied with folate in the circulation. The FR-alpha isfrequently overexpressed on a wide variety of specific cell types, suchas epithelial tumours (e.g. ovarian, cervical, endometrial, breast,colorectal, kidney, lung, nasopharyngeal), whereas the FR-beta isfrequently overexpressed in leukaemia cells (approx. 70% of acutemyelogenous leukaemia (AML) are FR-beta positive). Both may therefore beused as a valuable tumour marker for selective tumour-targeting (Elnakatand Ratnam, Adv. Drug Deliv. Rev. 2004; 56:1067-84). In addition it hasrecently been discovered that activated (but not resting) synovialmacrophages in patients diagnosed with rheumatoid arthritis possess afunctionally active FR-beta (Nakashima-Matsushita et al, Arthritis &Rheumatism, 1999, 42(8): 1609-16). Therefore activated macrophages canbe selectively targeted with folate conjugates in arthritic joints, acapability that opens possibilities for the diagnosis and treatment ofrheumatoid arthritis (Paulos et al, Adv. Drug Deliv. Rev. 2004;56:1205-17).

Folates is used herein as a generic term for a family ofchemically-similar compounds involved in a range of biosyntheticpathways. Folates consist of three units, which include (i) a condensedpyrimidine heterocycle unit, which is linked via a methylene group atthe C-6 position to (ii) a p-aminobenzoic acid unit, which is linked to(iii) one or more amino acid units. For example, in the case of folicacid derivatives, a pteridine heterocycle unit is linked via a methylenegroup at the C-6 position to a p-aminobenzoic acid unit, which is linkedto a variable number of glutamic acid units. Each of those three unitsmay be subjected to variation to create a library of various folatestructures. Such variations may include folates, that differ in theoxidation state of the pteridine ring, the type of the one carbonsubstituent at N5 and/or N10 positions, the type and number ofconjugated amino acid residues, and the substitution pattern of thevarious units. Folic acid itself as a synthetic analogue and member ofthe group of folates is the most oxidized form, whereas dihydrofolateand tetrahydrofolate are progressively more reduced forms of folates (astheir name indicates).

Folates are involved in the transfer of 1-C units in key syntheticpathways of bio-molecules such as methionine, purine, and pyrimidinebiosynthesis. Additionally, they play an important role in theinterconversion of serine and glycine, and in histidine catabolism.Folates and its derivatives have thus been intensively studied over thepast 15 years as targeting agents for the delivery of therapeutic and/ordiagnostic agents to cell populations bearing folate receptors in orderto achieve a selective concentration of therapeutic and/or diagnosticagents in such cells relative to normal cells.

Various probes have been conjugated to folic acid and (pre)clinicallyevaluated, including folate radiopharmaceuticals (Leamon and Low, DrugDiscov. Today 2001; 6:44-51 and Jammaz et al, J. Label Compd Radiopharm2006; 49:125-137), folate-conjugates of chemotherapeutic agents (Leamonand Reddy, Adv. Drug Deliv. Rev. 2004; 56:1127-41; Leamon et al,Bioconjugate Chem. 2005; 16:803-11), proteins and protein toxins (Wardet al, J. Drug Target. 2000; 8:119-23; Leamon et al, J. Biol. Chem.1993; 268:24847-54; Leamon and Low, J. Drug Target. 1994; 2:101-12),antisense oligonucleotides (Li et al, Pharm. Res. 1998; 15:1540-45; Zhaoand Lee, Adv. Drug Deliv. Rev. 2004; 56:1193-204), liposomes (Lee andLow, Biochim. Biophys. Acta-Biomembr. 1995; 1233:134-44; Gabizon et al,Adv. Drug Deliv. Rev. 2004; 56:1177-92), hapten molecules (Paulos et al,Adv. Drug Deliv. Rev. 2004; 56:1205-17), MRI contrast agents (Konda etal, Magn. Reson. Mat. Phys. Biol. Med. 2001; 12:104-13) etc. Typicallyall of these probes are conjugated to folic acid through its glutamateportion which lends itself to known carboxylic acid couplingmethodology.

Folate radiopharmaceuticals can be in particular very useful for animproved diagnosis and evaluation of the effectiveness of cancertherapy. This may include assessment and/or prediction of a treatmentresponse and consequently improvement of radiation dosimetry. Typicalvisualization techniques suitable for radioimaging are known in the artand include positron emission tomography (PET), planar or single photonemission computerized tomography (SPECT) imaging, gamma cameras,scintillation, and the like.

Both PET and SPECT use radiotracers to image, map and measure activitiesof target sites of choice. Yet, while PET uses positron emittingnuclides which require a nearby cyclotron due to the short half-lives ofthe positron emitters, SPECT uses single photon emitting nuclides whichare available by generator systems, which may make its use independentof nearby facilities such as cyclotrons or reactors and thus moreconvenient. However SPECT provides less sensitivity than PET and besidesa few approaches quantification methods are lacking. In contrast, PETPET shows a higher sensitivity (more than 100-fold of SPECT) andprovides well-elaborated quantification methods. Moreover, PET is one ofthe most sophisticated functional imaging technologies to assessregional uptake and affinity of ligands or metabolic substrates in brainand other organs and thus provides measures of imaging based onmetabolic activity. This is for example achieved by administering apositron emitting nuclide to a subject, and as it undergoes radioactivedecay the gamma rays resulting from the positron annihilation aredetected in the PET scanner by a ring of detectors which arecoincidentally connected in pairs.

Factors that need to be considered in the selection of a suitableisotope useful for PET include sufficient half-life of thepositron-emitting isotope to permit preparation of a diagnosticcomposition optionally in a pharmaceutically acceptable carrier prior toadministration to the patient, and sufficient remaining half-life toyield sufficient activity to permit extra-corporeal measurement by a PETscan. Furthermore, a suitable isotope should have a sufficiently shorthalf-life to limit patient exposure to unnecessary radiation. Typically,a suitable radiopharmaceutical for PET may be based on a metal isotope,such as gallium or copper. These two require however a chelator forentrapment of the metal, which may have an effect on steric and chemicalproperties. Alternatively a radiopharmaceutical may be based on acovalently linked isotope which provides minimal structural alteration.Radionuclides used for covalent attachment and suitable for PET scanningare typically positron emitting isotopes with short half lives such as¹¹C (ca. 20 min), ¹³N (ca. 10 min), ¹⁵O (ca. 2 min) and ¹⁸F (ca. 110min).

To date, a number of chelate-based folate radiopharmaceuticals have beensynthesized and successfully evaluated as diagnostic agents for imagingfolate receptor-positive tumors. The most widely studied derivativeswere labeled either with ¹¹¹In and ^(99m)Tc (Siegel et al., J. Nucl.Med. 2003, 44:700; Müller et al., J. Organomet. Chem. 2004, 689:4712) orwith ⁶⁸Ga (Mathias et al., Nucl. Med. Biol. 2003, 30(7):725). Yet onlythe latter one is a positron emitter and is suitable for PET imagingwhile the two former ones are single photon emitters and used for SPECT.Also all of the above need a suitable chelating agent, which istypically linked to folic acid through its amino acid, i.e. glutamateportion.

Thus a folate radiopharmaceutical having a covalently linked positronemitting nuclide would be of great interest. In particular a ¹⁸F-labeledfolate radiopharmaceutical would be most suitable for PET imagingbecause of its excellent imaging characteristics which would fulfill allof the above considerations. Compared with other suitable radionuclides(¹¹C, ¹³N, 150), ¹⁸F is very useful because of its longer half-life ofapproximately 110 minutes and because it decays by emitting positronshaving a low positron energy of 635 keV, which allows a veryhigh-resolution for PET images. Furthermore, the longer half-life of ¹⁸Falso allows for syntheses that are more complex and satellitedistribution to PET centers with no cyclotron and/or no radiochemistryfacilities. In addition the atomic radius of fluorine is comparable tothat of H. This implies that steric effects of a fluorine-for-Hsubstitution will hardly interfere with the binding of the ligand to thereceptor. Only the high electronegativity of fluorine may influence thebiochemical properties of a fluorinated ligand compared to theunsubstituted analogue.

Yet, the structure of folates does not lend itself to directradiolabeling with ¹⁵F. Thus to date, there have been only very few¹⁸F-labeled folates reported in the literature (Bettio et al., J. Nucl.Med., 2006, 47(7), 1153; WO 2006/071754). Moreover, these suggest¹⁸F-labeling through conjugation at the glutamate portion of folates. Todate there is no known ¹⁸F-labeled folate or derivative thereof, whereinthe fluorine-18 is linked within the folate skeleton, such as to thebenzoylamine moiety. In addition, the currently reported radiosynthesiswas time-consuming and gave only low radiochemical yields of less than5% (Bettio et al., J. Nucl. Med., 2006, 47(7), 1153) and thus isunsuitable for routine clinical applications.

Thus currently known ¹⁸F-labeled folates or derivatives thereof are notable to fill the need for specific radiopharmaceuticals suitable formetabolic imaging of tumors to improve diagnosis and treatment of cancerand inflammatory and autoimmune diseases.

Applicants have now found that ¹⁸F-labeled folate radiopharmaceuticalswherein the fluorine-18 is linked to the aminobenzoyl moiety within thefolate skeleton may be obtained through for example directradiolabeling.

Thus, the present invention is directed to new ¹⁸F-folateradiopharmaceuticals, wherein the fluorine-18 is covalently linked tothe aminobenzoyl moiety which links the pteridine heterocycle to theamino acid portion within folate structures, as well as theirprecursors, a method of their preparation, preferably through directradiolabeling, as well as their use in diagnosis and monitoring ofcancer or inflammatory and autoimmune disease therapy.

SUMMARY OF THE INVENTION

The present invention is in a first aspect directed to new ¹⁸F-folateradiopharmaceuticals and precursors thereof (hereinafter also calledcompounds of the invention), wherein the fluorine-18 and/or at least oneelectron-withdrawing group is covalently linked to the aminobenzoylmoiety.

In one specific embodiment, the new folate radiopharmaceuticals aresubstituted with the fluorine-18 in the 2′- and/or 6′-position of theaminobenzoyl-moiety, optionally comprising at least one furtherelectron-withdrawing group.

In a preferred embodiment the present invention is also directed towards2′- and 6′-¹⁸F-folate radiopharmaceuticals, optionally comprising atleast one further electron-withdrawing group.

In another specific embodiment, the present invention is directedtowards the precursors of the new folate radiopharmaceuticals. Theseinclude, for example, compounds that are substituted at the benzoylmoiety with at least one electron-withdrawing group which may act as aleaving group and is able to undergo nucleophilic aromatic substitutionby [¹⁸F]fluoride. Alternatively, the at least one electron-withdrawinggroup may act as an activator aiding the substitution by [¹⁸F]fluoride.Preferred electron-withdrawing groups may include for example nitro,cyano, (trimethyl)ammonium, sulfonates, esters, ketones, chloro, bromo,fluoro, iodonium salts, dialkyl/-aryl silanes, silanols and the like.

In a further aspect the present invention is directed to a method oftheir preparation. In a preferred embodiment the ¹⁸F-folateradiopharmaceuticals of the invention are obtained through direct¹⁸F-radiolabeling of suitable precursors.

In another aspect the present invention is directed to the use indiagnosis of a cell or population of cells expressing a folate-receptorand monitoring of cancer and cancer therapy in vitro or in vivo ormonitoring of inflammatory and autoimmune diseases such rheumatoidarthritis and therapy thereof.

In one embodiment, the present invention is directed towards uses of¹⁸F-folate radiopharmaceuticals of the invention for diagnostic imagingof a cell or population of cells expressing a folate-receptor.

More specifically the present invention includes methods for diagnosticimaging of a cell or population of cells expressing a folate-receptor,which includes for example methods for in vitro detection of a cellexpressing the folate receptor, for example a tumor cell or an activatedmacrophage, in a tissue sample. Such methods may also be performed invivo.

Thus, in a further embodiment the present invention is directed towardsuses of ¹⁸F-folate radiopharmaceuticals of the invention for convenientand effective administration to a subject in need for diagnostic imagingand/or monitoring of cancer or inflammatory and autoimmune diseasetherapy. The subject of the methods of the present invention ispreferably a mammal, such as an animal or a human, preferably a human.

Such methods of the invention may be performed in combination with anyother methods of diagnosis or therapy of cancer or inflammatory andautoimmune diseases including methods using other already developeddiagnostic and/or therapeutic agents and utilizing x-ray computedtomography (CT), magnetic resonance imaging (MRI), functional magneticresonance imaging (fMRI), single photon emission computed tomography(SPECT), optical imaging, and ultrasound.

Other features and advantages of the invention will be apparent from thefollowing detailed description thereof and from the claims.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Data from ex vivo biodistribution studies using2′-[¹⁸F]fluoro-folic acid: specific uptake in folate receptor-positivetissues.

FIG. 2. PET images using 2′-[¹⁸F]fluoro-folic acid (the arrows indicatethe position of the KB xenografts tumors).

FIG. 3. PET images using 2′-[¹⁸F]fluoro-folic acid (the arrows indicatethe kidneys).

FIG. 4. Ex vivo PET images of KB xenografts tumors using2′-[¹⁸F]fluoro-folic acid.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is in a first aspect directed to new ¹⁸F-folateradiopharmaceuticals and precursors thereof (hereinafter also calledcompounds of the invention), wherein the fluorine-18 and/or at least oneelectron-withdrawing group is covalently linked to the aminobenzoylmoiety.

¹⁸F is usually available as electrophilic [¹⁸F]F₂ and as generally usedherein, as nucleophilic [¹⁸F]fluoride. In form of [¹⁸F]fluoridefluorine-18 is producible more efficiently. In addition, this is theonly possibility for preparing no carrier added radiotracerssufficiently.

In a preferred embodiment a folate (structure) or derivative thereof,also hereinafter simply referred to as “a folate” or “folates”, for usein the present invention comprises compounds based on a condensedpyrimidine heterocycle, which is linked to an aminobenzoyl moietycarrying in para-position an amino acid portion. As used herein a“condensed pyrimidine heterocycle” includes a pyrimidine fused with afurther 5- or 6-membered heterocycle, such as a pteridine or apyrrolopyrimidine bicycle. As used herein the term “amino acid” includescompounds with both an amino group (e.g., NH₂ or NH₃ ⁺) and a carboxylicacid group (e.g., COOH or COO). In a specific embodiment, the amino acidmay be an α-amino acid, a β-amino acid, a D-amino acid or an L-aminoacid. The amino acid may be a naturally occurring amino acid (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine,asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine,or histidine, etc.) or it may be a derivative thereof. Examples ofderivatives include optionally substituted amino acids, e.g. having oneor more substituents selected from CN, Hal, and/or NO₂ (e.g.fluoroglutamic acid). The amino acid may also include any othernon-naturally occurring amino acids, such as e.g. norleucine, norvaline,L- or D-naphthalanine, ornithine, homoarginine and others well known inthe peptide art (see for example in M. Bodanzsky, “Principles of PeptideSynthesis,” 1st and 2nd revised ed., Springer-Verlag, New York, N.Y.,1984 and 1993, and Stewart and Young, “Solid Phase Peptide Synthesis,”2nd ed., Pierce Chemical Co., Rockford, Ill., 1984, both of which areincorporated herein by reference). Amino acids and amino acidanalogs/derivatives can be purchased commercially (Sigma Chemical Co.;Advanced Chemtech) or synthesized using methods known in the art. Inanother specific embodiment, the amino acid may also be part of apolyamino acid (also termed polypeptide), wherein a plurality of same ordifferent amino acids as defined hereinabove are covalently linked, i.e.linked through conventional peptide or other bonds. Preferred aminoacids include for example glutamic acid, aspartic acid, glutamine,aspartine, lysine, arginine, cystein, and derivatives thereof andpreferred polyamino acids include homopolymers the respectivehomopolymers thereof (i.e. polyglutamic acid, polyaspartic acid, etc).Most preferred are optionally substituted aspartic and glutamic acid.

Preferred representatives of folates as used herein are based on afolate skeleton, i.e. pteroyl-glutamic acid orN-[4(pteridin-6-ylmethylamino)benzoyl]-glutamic acid), and derivativesthereof and includes optionally substituted folic acid, folinic acid,pteropolyglutamic acid, and folate receptor-binding pteridines such astetrahydropterins, dihydrofolates, tetrahydrofolates, and their deazaand dideaza analogs. Folic acid is the preferred basic structure usedfor the compounds of this invention. The terms “deaza” and “dideaza”analogs refers to the art recognized analogs having a carbon atomsubstituted for one or two nitrogen atoms in the naturally occurringfolic acid structure. For example, the deaza analogs include the1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs. The dideazaanalogs include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza,and 5,8-dideaza analogs. Preferred deaza analogs compounds includeN-[4-[2-[(6R)-2-amino-1,4,5,6,7,8-hexahydro-4-oxopyrido[2,3-d]pyrimidin-6-yl]ethyl]benzoyl]-L-glutamicacid (Lometrexol) andN-[4-[1-[(2,4-diamino-6-pteridinyl)methyl]propyl]benzoyl]-L-glutamicacid (Edatrexate).

In a particular embodiment, the new folate radiopharmaceuticals arelabeled with the fluorine-18 in the 2′-, 3′-, 5′- or 6′-position of theaminobenzoyl-moiety, preferably in the 2′- or 6′-position. Mostpreferred are 2′- and 6′-¹⁸F-folate folate radiopharmaceuticals.Optionally the new folate radiopharmaceuticals further comprise at leastone electron-withdrawing group.

In another particular embodiment the present invention is directedtowards the precursors of these new folate radiopharmaceuticals, whereinthe aminobenzoyl moiety is substituted with at least oneelectron-withdrawing group, preferably selected from —NO₂, —CN,—N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts —I⁺(R′)₂,dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂, wherein R′ isindependently a straight-chain or branched C₍₁₋₁₂₎ alkyl group or anoptionally substituted carbocyclic and heterocyclic group comprisingfive-, six- or ten-membered ring systems most preferably one or twoelectron-withdrawing groups in the 2′- and/or 6′-position of theaminobenzoyl moiety.

Thus in a specific embodiment the present invention is directed towardscompounds of formula I,

wherein

-   A is an amino acid,-   X₁ to X₅ are independently of each other N or C,-   X₆, X₇ are independently of each other C, N or O,-   Z is an electron-withdrawing group preferably selected from —NO₂,    —CN, —N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts    —I⁺(R′)₂, dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiHR(″)₂,    wherein R′ is independently a straight-chain or branched C₍₁₋₁₂₎    alkyl group or an optionally substituted carbocyclic and    heterocyclic group comprising five-, six- or ten-membered ring    systems,-   R₁, R₂ are independently of each other H, Hal, —OR″, —NHR″, C1-C12    alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C2-C12 alkenyl, C2-C12    alkynyl, (C1-C12 alkoxy)carbonyl, and (C1-C12 alkylamino)carbonyl,    wherein R″ is H or C1-C6 alkyl,-   R₃, R₄ are independently of each other H, formyl, iminomethyl,    nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl,    halosubstituted C1-C12 alkanoyl,-   R₅, R₆ are independently of each other H or straight chain or    branched C₁-C₁₂ alkyl, which is unsubstituted or substituted by at    least one CN, Hal, or NO₂, and wherein one or more of embedded,    non-adjacent CH2 groups may independently be replaced by —O—, —CO—,    —CO—O—, —CO—NR′—, —CH═CH—, —C≡C—,-   m is 0, 1, 2 or 3,-   n is 0 or 1,-   p is 0, 1 or 2,-   q has a value of 1 to 7, and-   r is 0 or 1.

In a specific embodiment A is an amino acid selected from glutamic acid,aspartic acid, glutamine, aspartine, lysine, arginine, cystein, andderivatives thereof or a polyamino acid selected from the respectivehomopolymers. In a preferred embodiment A is optionally substitutedaspartic acid, glutamic acid, polyaspartic acid or polyglutamic acid.

Thus the present invention is further directed towards compounds offormula I wherein A is e.g. a glutamic acid residue, having formula II,

wherein

-   X₁ to X₅ are independently of each other N or C,-   X₆, X₇ are independently of each other C, N or O,-   Z is an electron-withdrawing group preferably selected from —NO₂,    —CN, —N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts    —I⁺(R′)₂, dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiHR(″)₂,    wherein R′ is independently a straight-chain or branched C₍₁₋₁₂₎    alkyl group or an optionally substituted carbocyclic and    heterocyclic group comprising five-, six- or ten-membered ring    systems,-   R₁, R₂ are independently of each other H, Hal, —OR″, —NHR″, C1-C12    alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C2-C12 alkenyl, C2-C12    alkynyl, (C1-C12 alkoxy)carbonyl, and (C1-C12 alkylamino)carbonyl,    wherein R″ is H or C1-C6 alkyl,-   R₃, R₄ are independently of each other H, formyl, iminomethyl,    nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl,    halosubstituted C1-C12 alkanoyl,-   R₅, R₆ are independently of each other H or straight chain or    branched C₁-C₁₂ alkyl, which is unsubstituted or substituted by at    least one CN, Hal, or NO₂, and wherein one or more of embedded,    non-adjacent CH2 groups may independently be replaced by —O—, —CO—,    —CO—O—, —CO—NR′—, —CH═CH—, —C≡C—,-   m is 0, 1, 2 or 3,-   n is 0 or 1,-   p is 0, 1 or 2,-   q has a value of 1 to 7, and-   r is 0 or 1.

In a preferred embodiment the fluorine-18 is at the 2′- or 6′-position.

In another preferred embodiment

R′ is H, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkanoyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, (C₁-C₆ alkoxy)carbonyl, or (C₁₋₆ alkylamino)carbonyl.

In a further preferred embodiment

R₃, R₄ are independently of each other H, formyl, iminomethyl, nitroso,C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, halosubstituted C1-C12alkanoyl, andR₅, R₆ are independently of each other H or straight chain or branchedC₁-C₁₂ alkyl, which is unsubstituted or substituted by at least one CN,Hal, or NO₂, and wherein one or more of embedded, non-adjacent CH2groups may independently be replaced by —O—, —CO—, —CO—O—, —CO—NR′—,—CH═CH—, —C≡C—.

In an even more preferred embodiment

-   R′ is H, methyl- or ethyl-,-   R₃, R₄ are independently of each other H, methyl- or formyl-, and-   R₅, R₆ are independently of each other H, methyl-, ethyl- or    tert.-butyl-.

More preferred are thus compounds of formulae III or IV,

wherein

-   X₁ to X₅ are independently of each other N or C,-   X₆, X₇ are independently of each other C, N or O,-   Z is an electron-withdrawing group preferably selected from —NO₂,    —CN, —N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts    —I⁺(R′)₂, dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂,    wherein R′ is independently a straight-chain or branched C₍₁₋₁₂₎    alkyl group or an optionally substituted carbocyclic and    heterocyclic group comprising five-, six- or ten-membered ring    systems,-   R₁, R₂ are independently of each other H, Hal, —OR″, —NHR″, C1-C12    alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C2-C12 alkenyl, C2-C12    alkynyl, (C1-C12 alkoxy)carbonyl, and (C1-C12 alkylamino)carbonyl,    wherein R″ is H or C1-C6 alkyl,-   R₃, R₄ are independently of each other H, formyl, iminomethyl,    nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl,    halosubstituted C1-C12 alkanoyl,-   R₅, R₆ are independently of each other H or straight chain or    branched C₁-C₁₂ alkyl, which is unsubstituted or substituted by at    least one CN, Hal, or NO₂, and wherein one or more of embedded,    non-adjacent CH2 groups may independently be replaced by —O—, —CO—,    —CO—O—, —CO—NR′—, —CH═CH—, —C≡C—,-   m is 0, 1, 2, or 3,-   p is 0, 1 or 2,-   q has a value of 1 to 7, and-   r is 0 or 1.

Preferred embodiments of compounds of formula I also apply to compoundsof formulae III and IV.

Further preferred compounds are compounds of formulae I, II, III or IV,wherein m=0. Thus, in another preferred embodiment, the presentinvention is directed towards compounds of formulae V and VI

wherein

-   X₁ to X₅ are independently of each other N or C,-   X₆, X₇ are independently of each other C, N or O,-   R₁, R₂ are independently of each other H, Hal, —OR″, —NHR″, C1-C12    alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C2-C12 alkenyl, C2-C12    alkynyl, (C1-C12 alkoxy)carbonyl, and (C1-C12 alkylamino)carbonyl,    wherein R″ is H or C1-C6 alkyl,-   R₃, R₄ are independently of each other H, formyl, iminomethyl,    nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl,    halosubstituted C1-C12 alkanoyl,-   R₅, R₆ are independently of each other H or straight chain or    branched C₁-C₁₂ alkyl, which is unsubstituted or substituted by at    least one CN, Hal, or NO₂, and wherein one or more of embedded,    non-adjacent CH2 groups may independently be replaced by —O—, —CO—,    —CO—O—, —CO—NR′—, —CH═CH—, —C≡C—,-   p is 0, 1 or 2,-   q has a value of 1 to 7, and-   r is 0 or 1.

Preferred embodiments of compounds of formulae I to IV also apply tocompounds of formulae V and VI.

In another specific embodiment, the present invention is directedtowards compounds of formula I wherein m is 1 or 2, such that morepreferably the electron-withdrawing group(s) Z is at the 2′- and/or6′-position.

More preferred are thus compounds of formulae VII, VIII, IX, X and XI,

wherein

-   X₁ to X₅ are independently of each other N or C,-   X₆, X₇ are independently of each other C, N or O,-   Z is a electron-withdrawing group preferably selected from —NO₂,    —CN, —N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts    —I⁺(R′)₂, dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂,    wherein R′ is independently a straight-chain or branched C₍₁₋₁₂₎    alkyl group or an optionally substituted carbocyclic and    heterocyclic group comprising five-, six- or ten-membered ring    systems,-   R₁, R₂ are independently of each other H, Hal, —OR″, —NHR″, C1-C12    alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C2-C12 alkenyl, C2-C12    alkynyl, (C1-C12 alkoxy)carbonyl, and (C1-C12 alkylamino)carbonyl,    wherein R″ is H or C1-C6 alkyl,-   R₃, R₄ are independently of each other H, formyl, iminomethyl,    nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl,    halosubstituted C1-C12 alkanoyl,-   R₅, R₆ are independently of each other H or straight chain or    branched C₁-C₁₂ alkyl, which is unsubstituted or substituted by at    least one CN, Hal, or NO₂, and wherein one or more of embedded,    non-adjacent CH2 groups may independently be replaced by —O—, —CO—,    —CO—O—, —CO—NR′—, —CH═CH—, —C≡C—,-   n is 0 or 1,-   p is 0 or 1,-   q has a value of 1 to 7, and-   r is 0 or 1.

Preferred embodiments of compounds of formulae I to VI also apply tocompounds of formulae VII, VIII, IX, X and XI.

Further preferred compounds are compounds of formulae I, VII, VIII, IX,X or XI, wherein n=0. Thus, in another preferred embodiment, the presentinvention is directed towards compounds of formulae XII, XIII and XIV

wherein

-   X₁ to X₅ are independently of each other N or C,-   X₆, X₇ are independently of each other C, N or O,-   Z is an electron-withdrawing group preferably selected from —NO₂,    —CN, —N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts    —I⁺(R′)₂, dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂,    wherein R′ is independently a straight-chain or branched C₍₁₋₁₂₎    alkyl group or an optionally substituted carbocyclic and    heterocyclic group comprising five-, six- or ten-membered ring    systems,-   R₁, R₂ are independently of each other H, Hal, —OR″, —NHR″, C1-C12    alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C2-C12 alkenyl, C2-C12    alkynyl, (C1-C12 alkoxy)carbonyl, and (C1-C12 alkylamino)carbonyl,    wherein R″ is H or C1-C6 alkyl,-   R₃, R₄ are independently of each other H, formyl, iminomethyl,    nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl,    halosubstituted C1-C12 alkanoyl,-   R₅, R₆ are independently of each other H or straight chain or    branched C₁-C₁₂ alkyl, which is unsubstituted or substituted by at    least one CN, Hal, or NO₂, and wherein one or more of embedded,    non-adjacent CH2 groups may independently be replaced by —O—, —CO—,    —CO—O—, —CO—NR′—, —CH═CH—, —C≡C—,-   p is 0, 1 or 2,-   q has a value of 1 to 7, and-   r is 0 or 1.

Preferred embodiments of compounds of formulae I to XI also apply tocompounds of formulae XII, XIII and XIV.

It is understood, that the abbreviations “N” and “C” are representativefor all possible degrees of saturation, i.e. N includes —NH— and —N═linkages and C includes —CH₂— and —CH═ linkages.

It is further understood, that (H)_(q) represents all H substituents onthe indicated ring (i.e. on X₃, C6, C7 and X₄). For example q=5 for afully saturated unsubstituted analog (X₃=X₄=N, p=0) or q=7 for a fullysaturated unsubstituted 5,8-dideaza analog (X₃=X₄=C, p=0) and q=1 for afully unsaturated analog with X₃=X₄=N, p=0.

A preferred embodiment of compounds of formulae I to XIV includes forexample wherein X₁ to X₅ are N, R₁ is NY₄Y₅, R₂ is O, p is 0 and q is 1.

Thus, in a further specific embodiment the present invention is directedto a compound of formula XV,

wherein

-   Z is an electron-withdrawing group preferably selected from —NO₂,    —CN, —N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts    —I⁺(R′)₂, dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂,    wherein R′ is independently a straight-chain or branched C₍₁₋₁₂₎    alkyl group or an optionally substituted carbocyclic and    heterocyclic group comprising five-, six- or ten-membered ring    systems,-   R₅, R₆, are independently of each other H or straight chain or    branched C₁-C₁₂ alkyl, which is unsubstituted or substituted by at    least one CN, Hal, or NO₂, and wherein one or more of embedded,    non-adjacent CH2 groups may independently be replaced by —O—, —CO—,    —CO—O—, —CO—NR′—, —CH═CH—, —C≡C—,-   Y₁, Y₂ are independently of each other selected from H, formyl,    straight chain or branched C₁-C₁₂ alkyl, which is unsubstituted or    substituted by at least one CN, Hal, or NO₂, and wherein one or more    of embedded, non-adjacent CH2 groups may independently be replaced    by —O—, —CO—, —CO—O—, —CO—NR′—, —CH═CH—, —C≡C—,-   R₄ is selected from H, nitroso, C₁-C₁₂ alkyl, C₁-C₁₂ alkoxy, C₁-C₁₂    alkanoyl, halosubstituted C₁-C₁₂ alkanoyl,-   m is 0, 1, 2 or 3, and-   n is 0 or 1.

Preferred embodiments of compounds of formulae I to XIV also apply tocompounds of formula XV.

Thus, in a further specific embodiment the present invention is directedto a compound of formula XVI,

wherein

-   Z is an electron-withdrawing group preferably selected from —NO₂,    —CN, —N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts    —I⁺(R′)₂, dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂,    wherein R′ is independently a straight-chain or branched C₍₁₋₁₂₎    alkyl group or an optionally substituted carbocyclic and    heterocyclic group comprising five-, six- or ten-membered ring    systems,-   R₃, R₄ are independently of each other H, formyl, iminomethyl,    nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl,    halosubstituted C1-C12 alkanoyl,-   R₅, R₆ are independently of each other H or straight chain or    branched C₁-C₁₂ alkyl, which is unsubstituted or substituted by at    least one CN, Hal, or NO₂, and wherein one or more of embedded,    non-adjacent CH2 groups may independently be replaced by —O—, —CO—,    —CO—O—, —CO—NR′—, —CH═CH—, —C≡C—,-   Y₁, Y₂ are independently of each other selected from H, formyl,    straight chain or branched C₁-C₁₂ alkyl, which is unsubstituted or    substituted by at least one CN, Hal, or NO₂, and wherein one or more    of embedded, non-adjacent CH2 groups may independently be replaced    by —O—, —CO—, —CO—O—, —CO—NR′—, —CH═CH—, —C≡C—,-   m is 0, 1, 2 or 3, and-   n is 0 or 1.

Preferred embodiments of compounds of formulae I to XV also apply tocompounds of formula XVI.

Other embodiments are compounds of formulae I to XIV wherein X₁ to X₅and R₁ and R₂ are N, R₃=R₅=R₆ is H, R₄ is CH₃, p is 0 and q is 1.

Thus, in a further specific embodiment the present invention is directedto a compound of formula XVII,

wherein

-   Z is an electron-withdrawing group preferably selected from —NO₂,    —CN, —N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts    —I⁺(R′)₂, dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂,    wherein R′ is independently a straight-chain or branched C₍₁₋₁₂₎    alkyl group or an optionally substituted carbocyclic and    heterocyclic group comprising five-, six- or ten-membered ring    systems,-   R₅, R₆ are independently of each other H or straight chain or    branched C₁-C₁₂ alkyl, which is unsubstituted or substituted by at    least one CN, Hal, or NO₂, and wherein one or more of embedded,    non-adjacent CH2 groups may independently be replaced by —O—, —CO—,    —CO—O—, —CO—NR′—, —CH═CH—, —C≡C—,-   m is 0, 1, 2 or 3, and-   n is 0 or 1.

Other embodiments are compounds of formulae I to XIV wherein X₁ to X₅and R₁ and R₂ are N, R₄=R₅=R₆ is H, R₃ is CH₃ or formyl, p is 1 and q is4.

Thus, in a further specific embodiment the present invention is directedto a compound of formula XVIII,

wherein

-   Z is an electron-withdrawing group preferably selected from —NO₂,    —CN, —N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts    —I⁺(R′)₂, dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂,    wherein R′ is independently a straight-chain or branched C₍₁₋₁₂₎    alkyl group or an optionally substituted carbocyclic and    heterocyclic group comprising five-, six- or ten-membered ring    systems,-   R₃ is H, methyl- or formyl-,-   R₅, R₆ are independently of each other H or straight chain or    branched C₁-C₁₂ alkyl, which is unsubstituted or substituted by at    least one CN, Hal, or NO₂, and wherein one or more of embedded,    non-adjacent CH2 groups may independently be replaced by —O—, —CO—,    —CO—O—, —CO—NR′—, —CH═CH—, —C≡C—,-   m is 0, 1, 2 or 3, and-   n is 0 or 1.

The term “alkyl”, when used singly or in combination, refers to straightchain or branched alkyl groups typically containing 1 to 12, preferably1 to 8 more preferably 1 to 4 carbon atoms, such as methyl, ethyl,propyl, isopropyl, butyl, sec-butyl, isobutyl, t-butyl, pentylisopentyl, neopentyl, hexyl and the like.

As used herein, the term “alkenyl” (i.e. an alkyl group as defined abovehaving at least one double bond), singly or in combination with othergroups, refers to straight chain or branched alkylene groups containing2 to 12 carbon atoms, such as methylene, ethylene, propylene,isopropylene, butylene, t-butylene, sec-butylene, isobutylene, amylene,isoamylene, pentylene, isopentylene, hexylene and the like. Thepreferred alkenyl groups contain 2 to 8 carbon atoms.

The term “alkynyl” (i.e. an alkyl group as defined above having at leastone triple bond) as used herein refers to a linear or branched chain ofcarbon atoms with one or more carbon-carbon triple bonds. The preferredalkynyl groups contain 2 to 12, more preferably 2 to 8 carbon atoms.

The term “alkoxy” as used herein refers to an alkyl, as defined above,substituted with oxygen, such as methoxy, ethoxy, propoxy, isopropoxy,butoxy, tert-butoxy and the like.

The term “alkanoyl” as used herein refers to formyl, or an alkyl, asdefined above, terminally-substituted with a carbonyl such as acetyl,propanoyl, butanoyl, pentanoyl and the like.

The term “alkylamino” as used herein refers to an alkyl, as definedabove, substituted with nitrogen, including both monoalkylamino such asmethylamino, ethylamino, propylamino, tertbutylamino, and the like, anddialkylamino such as dimethylamino, diethylamino, methylpropylamino, andthe like.

The term “halo” as used herein refers to any Group 17 element andincludes fluoro, chloro, bromo, iodo, and astatine(o).

The expression “optionally substituted” preferably includes substitutionwith hydroxy, alkoxy, (di)alkylamino, alkylsulfonyl, alkylcarbonyl,alkylcarbonyloxy, alkoxycarbonyl, carboxyl, Hal, CN, NO₂.

The expression “carbocyclic and heterocyclic group comprising five-,six- or ten-membered ring systems and the like” preferably includesphenyl, naphthyl, azetidinyl, pyrrolidinyl, imidazolyl, indolyl,oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, oxatriazolyl,thiatriazolyl, pyridazinyl, morpholinyl, pyrimidinyl, pyrazinyl,pyridyl, quinolinyl, isoquinolinyl, piperidinyl, pyrazolyl,imidazopyridinyl and piperazinyl, more preferably phenyl, naphthyl,pyrrolidinyl, imidazolyl, triazolyl, pyrimidinyl, pyridyl, piperidinyl,and pyrazolyl, most preferably phenyl, pyridyl and naphthyl.

The term “electron-withdrawing group” or “group Z” as used herein refersto a functionality, which can act as a leaving group and thus can beexchanged by an incoming [¹⁸F]fluoride or else can act as an activatorfor the introduction of the [¹⁸F]fluoride. Suitable electron-withdrawinggroups include —NO₂, —CN, —N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br,—F, iodonium salts —I⁺(R′)₂, dialkyl/-aryl silanes —SiOH(R′)₂, andsilanols —SiH(R′)₂, wherein R′ is independently a straight-chain orbranched C₍₁₋₁₂₎ alkyl group or an optionally substituted carbocyclicand heterocyclic group comprising five-, six- or ten-membered ringsystems and the like, preferably —NO₂, —CN, —N⁺(CH₃)₃, —SO₃R′, —COOR′,—COR′, —Cl, —Br, —F, more preferably —NO₂, —CN, —N⁺(CH₃)₃.

In a preferred embodiment R₁ and R₂ are independently of each other H,—OR″, —NHR″ wherein R″ is H, C₁-C₆ alkyl, C₁-C₄ alkoxy, C₁-C₄ alkanoyl,(C₁-C₄ alkoxy)carbonyl, and (C₁-C₆ alkylamino)carbonyl, more preferablyR₁ and R₂ are independently of each other —OH, NH₂.

In a preferred embodiment R₃ and R₄ are independently of each other H,methyl or formyl.

In a preferred embodiment R₅ and R₆ are independently of each other H,methyl, ethyl or tert.-butyl.

In a preferred embodiment R′ is H, methyl or ethyl.

In a preferred embodiment R″ is H, methyl or ethyl.

In a further aspect the present invention provides a method ofsynthesizing a compound of the invention. Applicants have found that thefolate radiopharmaceuticals of the invention may be obtained throughdirect radiolabeling with [¹⁸F]fluoride.

More specifically, a method of production of the invention comprises thesteps of providing a precursor having formula I wherein n=0 and reactingsaid precursor with [¹⁸F]fluoride activated by phase transfer catalystssuch as tetrabutylammonium carbonate or aminopolyethers (e.g. Kryptofix©2.2.2) in combination with potassium carbonate or oxalate to form acompound having formula I including a [¹⁸F]fluoro group. In a preferredembodiment the folate radiopharmaceuticals were obtained in a directlabeling method based on a fluoro-for-nitro-exchange.

In a typical reaction, a suitable organic solvent was added to dry¹⁸F-Fluoride-cryptate and the resulting solution was added to a suitablyprotected precursor, which was provided in a sealed reaction vessel witha base such as DIEA, TEA or pyridine. The resulting mixture was heatedto 140-145° C. for 20-25 min. After short cartridge purification,deprotection was carried out under basic or acidic conditions and agentle heating for 5-10 min. Crude product solution was neutralized andinjected to semi-prep HPLC system. The radioactive product was collectedand the HPLC solvents removed by a stream of nitrogen, vacuum and gentleheating. For the formulation, the dry product was redissolved withphysiological solution and transferred to sterile vial using a sterilefilter.

In a further aspect the present invention provides uses of folateradiopharmaceuticals of the invention for convenient and effectiveadministration to a subject in need for diagnostic imaging.

Thus the present invention provides a method for diagnostic imaging of acell or population of cells expressing a folate-receptor, said methodcomprising the steps of administering at least one folateradiopharmaceutical of the invention in a diagnostic imaging amount, andobtaining a diagnostic image of said cell or population of cells.

Such imaging may be performed on a cell or population of cellsexpressing a folate-receptor in vitro or in vivo.

Thus, the present invention provides a method for in vitro detection ofa cell expressing the folate receptor in a tissue sample which includescontacting said tissue sample with at least one folateradiopharmaceutical of the invention in effective amounts and forsufficient time and conditions to allow binding to occur and detectingsuch binding by imaging techniques such as autoradiography and the like.

In a further aspect the present invention provides uses of folateradiopharmaceuticals of the present invention for convenient andeffective administration to a subject in need for diagnostic imaging ormonitoring of cancer or inflammatory and autoimmune disease therapy.

In another aspect the present invention provides a method forsimultaneous diagnosis and therapy, comprising the steps ofadministering to a subject in need thereof at least one folateradiopharmaceutical of the present invention in a diagnosticallyeffective amount in combination with a therapeutically active, andobtaining a diagnostic image of said tissues to follow the course oftreatment.

The subject of the methods of the present invention is preferably amammal, such as an animal or a human, preferably a human.

The dosage depends on the nature of the effect desired, such as the formof diagnosis or therapy, on the kind and frequency of treatment, on thediagnostic instrumentation, on the form of application of thepreparation, and on the age, weight, nutrition and condition of therecipient, kind of concurrent treatment, if any.

However, the most preferred dosage can be tailored to the individualsubject, as is understood and determinable by one of skill in the art,without undue experimentation. This typically involves adjustment of astandard dose, e.g., reduction of the dose if the patient has a low bodyweight.

Treatment can commence with a smaller amount, below the optimum amount,which can be increased in order to achieve the optimum effect.

The folate radiopharmaceuticals of the present invention may beadministered either as a repeated dose or preferably as a single dose.For example, the folate radiopharmaceuticals of this invention may beadministered to a subject by intravenous bolus injection. The suitableforms for injection include sterile aqueous solutions or dispersions ofthe above mentioned folate radiopharmaceuticals of the presentinvention.

For a solution to be injected a preferred unit dosage is from about 0.01mL to about 10 mL. After e.g. intravenous administration, imaging of theorgan or tumor in vivo can take place, if desired, from 30 min to 4hours, after the radiolabeled reagent has been administered to asubject. Typically, a sufficient amount of the administered dose willaccumulate in the targeted area.

The folate radiopharmaceuticals are preferably purified by HPLC. Afterremoving the solvents of the HPLC purification the products werepreferably solved in physiological solutions such as 0.9% NaCl or 0.15Mphosphate buffer solution, before the application, the formulatedradiopharmaceutical is transferred to a sterile vial via a sterilefilter.

The folate radiopharmaceuticals of the invention may also be used for invitro detection of a cell expressing the folate receptor in a tissuebiopsy taken from a subject. Thus in a further embodiment the presentinvention provides a method for in vitro detection of a cell expressingthe folate receptor, e.g. a tumor cell or an activated macrophage, in atissue sample which includes contacting said tissue sample with a folateradiopharmaceutical of the present invention in effective amounts andfor sufficient time and conditions to allow binding to occur anddetecting such binding by imaging techniques.

Samples can be collected by procedures known to the skilled person,e.g., by collecting a tissue biopsy or a body fluid, by aspirating fortracheal or pulmonary samples and the like.

Tissue samples to be tested include any tissue suspected to contain acell expressing a folate receptor, such as tumor cells, epithelialcells, kidneys, gastrointestinal or the hepatobiliary system, activatedmacrophages, monocytes, and others. Samples can be sectioned, e.g., witha microtome, to facilitate microscopic examination and observation.Samples can also be fixed with an appropriate fixative either before orafter incubation with one of the folate radiopharmaceuticals of thepresent invention to improve the histological quality of sample tissues.

Time and conditions sufficient for binding of a folateradiopharmaceutical of the present invention to a folate receptor on thecell include standard tissue culture conditions, i.e. samples can becultured in vitro and incubated with one of the compounds orcompositions of the present invention in physiological media. Suchconditions are well known to the skilled person. Alternatively, samplescan be fixed and then incubated with a folate radiopharmaceutical of thepresent invention in an isotonic or physiological buffer.

For all applications it is convenient to prepare the compounds orcompositions of the present invention at, or near, the site where theyare to be used.

All of the compounds and/or methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. It will be apparent to those of skill in the art thatvariations may be applied to the present invention without departingfrom the scope of the invention. The Examples provided herein areintended to be illustrative and are not exhaustive; therefore theillustrated Examples should not be viewed as limiting the invention inany way.

EXAMPLES Materials and Methods

Production of [¹⁸F]fluoride n.c.a. [¹⁸F]fluoride was produced via the¹⁸O (p,n)¹⁸F nuclear reaction at a Cyclone 18/9 cyclotron (IBA,Belgium). Isotopically 97% enriched [¹⁸O]water was irradiated by a 16MeV proton beam using a 2.1 ml liquid target. The[¹⁸F]fluoride/[¹⁸O]water solution was transferred from the target to amanipulator equipped syntheses hotcell using a helium stream.[¹⁸F]fluoride (˜20-30 GBq) was trapped on an anion exchange cartridge(Sep-Pak® Light Accell Plus QMA, Waters AG), preconditioned with 5 ml0.5M potassium carbonate solution and 5 ml water, while the [¹⁸O]waterwas recovered for recycling. ¹H-NMR-spectra: ¹H-NMR-spectra wererecorded on a Varian Mercury Plus 200 (200 MHz) spectrometer. Chemicalshifts were reported using TMS (Tetramethylsilan) as an internalstandard. The electron spray ionisation mass spectra were recorded on anAgilent XCT spectrometer.

HPLC: For HPLC analysis of the precursors and the 2′-fluorofolic acidthe following HPLC method was used: eluent A was aq. 0.05 M NaH₂PO₄which was adjusted to pH 7.0 by addition of 32% aq. sodium hydroxidesolution. Eluent B was a 1:1 mixture of solvent A and methanol. Thecolumn used was RP 18, Nucelosil, the gradient was from 100% eluent A to100% eluent B within 30 min., 20 mg of the sample were dissolved in abuffer consisting of 20 g NaHCO₃ and 20 g KHCO₃ in 1000 ml of water.

For all other intermediates the following HPLC method was used: Samemethod as described above, but eluent B was composed of 800 ml methanoland 200 ml 0.05 M NaH₂PO₄.

For semi-preparative HPLC purification of the 2′[¹⁸F]fluorofolic acidwas carried out on a RP 18 column, Gemini 5μ C18, 250×10 mm, using agradient as follows. Solvent A=0.05M phosphate buffer solution,B=methanol, 0-30 min: A: 99%→40%, 30-40 min: A: 40%→10%, 40-45 min: A:40%→99%.

Example 1 Synthesis of 2′-nitrofolic acid (a) Synthesis of4-(tert-butoxycarbonylamino)-2-nitrobenzoic acid

To a suspension of 1 g of 4-amino-2-nitro-benzoic acid in 10 ml of water0.69 g of aqueous sodium hydroxide solution (32%) were added followed bya solution of 1.2 g di-tert.-butyl-dicarbonate in 12 ml of dioxane.After 29 hours at room temperature additional 0.24 g ofdi-tert.-butyl-dicarbonate were added and the mixture was stirred forfurther 2 hours at room temperature. The reaction mixture was washedthree times with methyl-tert.butylether. The aqueous layer was treatedwith a 10% aqueous solution of citric acid until pH=3 was obtained. Theresulting suspension was cooled to 0° C. The product was sucked off,washed with water and dried at 40° C. under vacuum to give 0.72 g of4-(tert-butoxycarbonylamino)-2-nitrobenzoic acid.

(b) Synthesis ofdi-tert-butyl-N-(4-(tert.-butoxycarbonylamino)-2-nitrobenzamido)-L-glutamate

To a mixture of 4-(tert-butoxycarbonylamino)-2-nitrobenzoic acid in 60ml of dichloromethane were added 4.8 g ofN,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-uronium-hexafluoro-phosphate.After stirring for 15 min. a mixture of 3.8 g of L-glutamic acid-ditert.-butylester hydrochloride in 60 ml dichloromethane and 3 mltriethylamin was added dropwise. After stirring for 20 hours at roomtemperature, the mixture was filtered and the filtrate was washed fivetimes with 10% aqueous citric acid, four times with 5% aqueous sodiumcarbonate solution and two times with water. The organic layer was driedover magnesium sulphate and concentrated in vacuum to give 6.3 g ofdi-tert-butylN-(4-(tert-butoxycarbonylamino)-2-nitrobenzamido)-L-glutamate as ayellow foam. This was directly used in example 3.

(c) N-(4-amino-2-nitrobenzamido)-L-glutamic acid×trifluoroacetic acidsalt

To a solution ofdi-tert.-butyl-N-(4-(tert-butoxycarbonylamino)-2-nitrobenzamido)-L-glutamatein 53 ml dichloromethane were added 53 ml of trifluoroacetic acid at 0°C. under argon. After 1 hour at room temperature the mixture wasconcentrated to dryness to give 3.54 g ofN-(4-amino-2-nitrobenzamido)-L-glutamic acid as a yellow foam.

(d) Synthesis of 2′-nitrofolic acid

To a solution of 3.5 g of N-(4-amino-2-nitrobenzamido)-L-glutamicacid×trifluoroacetic acid salt in 50 ml of dimethylacetamide 2.31 g of2-amino-4-oxo-6-brommethyl-pteridine hydrobromide were added undernitrogen atmosphere. The suspension was stirred at 60° C. for 5 hoursand then for 20 hours at room temperature. Solids were removed byfiltration and washed with dimethylacetamide. The filtrate was addeddropwise within 10 min. to 321 ml of 0.1 M aqueous hydrochloric acid atroom temperature. The resulting suspension was stirred for 2 hours atroom temperature. The product was sucked off, washed with 24 ml of 0.1 Maqueous hydrochloric acid, 24 ml of water, dried at 40° C. under vacuumto give 1.54 g of crude 2′-nitrofolic acid which was purified byrecrystallization from water to give 1.04 g of pure 2′-nitrofolic acid.(HPLC purity: 98.1% area, m/z=487 [M+1]⁺, ¹H-NMR (200 MHz, DMSO-d₆)[ppm]: 8.65 (s, C(7)-H, 1H); 7.75 (t, N(8′)-H, 1H, exchangeable withD₂O); 7.51 (t, C(3′H), 1H); 7.20 (t, N(10)-H, 1H, exchangeable withD₂O); 7.03 (bs, NH₂, 2H, exchangeable with D₂O); 6.50 (m, C(5′)-H,(C(6′)-H, 2H); 4.48 (d, C(6)H₂, 2H); 4.29 (m, C(α)-H, 1H); 2.28 (m,C(β)-H₂, 2H); 1.96 (m, C(γ)-H₂, 2H).

Example 2 Synthesis of 2′-nitrofolic acid dimethyl esterbenzenesulfonate

Esterification of 2′nitrofolic acid was achieved in analogy to themethod described for esterification of folic acid in WO 2001/04121.

Example 3 Synthesis of N²,N,N-dimethylaminomethylene-2′-nitrofolicacid-di-tert. butylester (a) Synthesis of4-(((9H-fluorene-9-yl)methoxy)carbonylamino)-2-nitrobenzoic acid

To a solution of 11.4 g 4-amino-2-nitro-benzoic acid in 228 ml of watercontaining 6.63 g of sodium carbonate were added 17.0 g of9-fluorenylmethyl-chloroformate and dropwise 20 ml of dioxane. Afterstirring for 20 hours under nitrogen, the mixture was filtered and thefiltrate was washed five times with methyl-tert.-butylether. Residualmethyl-tert.-butylether was removed from the aqueous phase byevaporation under vacuum. To the aqueous phase were added 456 g of 0° C.cold water. The mixture was adjusted to pH=3 by addition of 31 ml of 2Maqueous hydrochloric acid. The precipitate was sucked off, washed with513 ml of water, dried at 40° C. in vacuum to give 17.0 g of4-(((9H-fluorene-9-yl)methoxy)carbonylamino)-2-nitrobenzoic acid asoff-white crystals.

(b) Synthesis ofdi-tert-butyl-N-(4-(((9H-fluorene-9-yl)methoxy)carbonylamino)-2-nitrobenzamido)-L-glutamate

To a suspension of 17.2 g4-(((9H-fluorene-9-yl)methoxy)carbonylamino)-2-nitrobenzoic acid in 222ml dichloromethane were added 17.7 g ofN,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-uronium-hexafluoro-phosphate.After stirring for 15 min. at room temperature a solution of 13.8 g ofL-glutamic acid-di-tert.butylester hydrochloride in 172 mldichloromethane and 12.9 ml triethylamine were added dropwise within 30min. The mixture was stirred under nitrogen at room temperature for 20hours. After addition of 860 ml of methyl-tert.-butylether the mixturewas washed five times with aqueous sodium H carbonate (5%), 5 times withaqueous citric acid (5%) and two times with brine. The organic layer wasdried over magnesium sulphate and evaporated to dryness under vacuum togive 27.9 g of crude di-tert-butyl2-(4-(((9H-fluorene-9-yl)methoxy)carbonylamino)-2-nitrobenzamido)-L-glutamateas a yellow foam. The crude product was purified by flash chromatographyusing silica gel 60 and ethylacetate/n-heptane/45:55 as eluent. Afterevaporation of product fractions 23.8 g ofdi-tert-butyl-N-(4-(((9H-fluorene-9-yl)methoxy)carbonylamino)-2-nitrobenzamido)-L-glutamatewere obtained as a yellow foam (HPLC, purity: 99.9% area).

(c) Synthesis of N-(4-amino-2-nitrobenzamido)-L-glutamic aciddi-tert.-butylester

To a mixture of 10 g of di-tert-butylN-(4-(((9H-fluorene-9-yl)methoxy)carbonylamino)-2-nitrobenzamido)-L-glutamatein 200 ml of N,N-dimethylformamide 1.34 ml pyrrolidine were added. Themixture was stirred for 30 min. at room temperature and then evaporatedto dryness under vacuum. After addition of 200 ml of diisopropyletherand stirring for 15 min., the resulting suspension was kept at 0° C.over night. The product was sucked-off, washed with 60 ml ofdiisopropylether and then dried under vacuum to give 4.3 g ofN-(4-amino-2-nitrobenzamido)-L-glutamic acid di-tert.-butylester asyellow needles (HPLC, assay 99.7% area).

(d) Synthesis of 2′-nitrofolic acid-di-tert.-butylester

To a solution of 1 g of N-(4-amino-2-nitrobenzamido)-L-glutamic aciddi-tert.-butylester in 100 ml of dimethylacetamide were added 2.1 g of2-amino-4-oxo-6-brommethyl-pteridine hydrobromide. The mixture wasstirred under nitrogen at 60° C. for 13 hours. After cooling to roomtemperature, the mixture was filtered and the filtrate was addeddropwise to 700 ml of water. The crystals were sucked off, washed with70 ml of water and dried at 35° C. under vacuum to give 1.12 g of2′-nitrofolic acid-di-tert.-butylester.

(e) Synthesis of N²,N,N-dimethylaminomethylene-2′-nitrofolicacid-di-tert.-butylester

To a solution of 1 g of 2′-nitrofolic acid-di-tert.-butylester in 150 mldry dimethylformamide were added 3.5 ml of diisopropyldimethylacetal.The mixture was stirred under nitrogen for 20 hours at room temperatureand was then evaporated to dryness. The residue was purified by flashchromatography using silica gel 60 and dichloromethane/methanol/95:5 aseluent to give 0.62 g of N²,N,N-dimethylaminomethylene-2′-nitrofolicacid-di-tert.-butylester.

Example 4 Synthesis of N²,N,N-dimethylaminomethylene-2′-nitrofolicacid-dimethyl ester (a) Synthesis ofdimethyl-N-(4-(tert.-butoxycarbonylamino)-2-nitrobenzamido)-L-glutamate

The synthesis was achieved in analogy to example 2 by using L-glutamicacid-dimethylester-hydrochloride instead of the L-glutamicacid-di-tert.-butylester-hydrochloride. From 14.8 g of4-(tert-butoxycarbonylamino)-2-nitrobenzoic acid 27.4 g of crudedimethyl-N-(4-(tert.-butoxycarbonylamino)-2-nitrobenzamido)-L-glutamatewere obtained which were purified twice by flash chromatography usingsilicagel 60 and ethylacetate/n-heptane/65:35 as eluent. Afterpurification 17.38 g ofdimethyl-N-(4-(tert.butoxycarbonylamino)-2-nitrobenzamido)-L-glutamatewere obtained.

(b) Synthesis of N-(4-amino-2-nitrobenzamido)-L-glutamicacid-dimethylester×trifluoroacetic acid salt

The synthesis was achieved in analogy to example 3. From 17 g ofdimethyl-N-(4-(tert.-butoxycarbonylamino)-2-nitrobenzamido)-L-glutamate19.5 g of N-(4-amino-2-nitrobenzamido)-L-glutamicacid-dimethylester×trifluoroacetic acid salt were obtained which wereused directly in example 4(c).

(c) Synthesis of 2′-nitrofolic acid dimethylester

The synthesis was performed in analogy to example 8 starting from 5 g ofN-(4-amino-2-nitrobenzamido)-L-glutamicacid-dimethylester×trifluoroacetic acid salt. The work-up was modifiedas follows. After filtration of the reaction mixture the filtrate wasadded dropwise to 3.5 l of 0.1 M aqueous hydrochloric acid. The mixturewas kept at 0° C. over night and the product was sucked-off, washed with50 ml of 0.1 M aqueous hydrochloric acid, 300 ml of water and then driedat 40° C. in vacuum to give 2.14 g of crude 2′-nitrofolic aciddimethylester. From the mother liquor further 0.82 g of crude2′-nitrofolic acid dimethylester were obtained. 2.9 g of crude2′-nitrofolic acid dimethylester were recrystallized from DMAC to give2.28 g of pure 2′-nitro folic acid dimethylester.

(d) Synthesis of N²,N,N-dimethylaminomethylene-2′-nitrofolicacid-dimethyl ester

This was done in analogy to example 3(e).

Example 5 Synthesis of 2′-fluorofolic acid

The synthesis was done in analogy to the synthesis of 2′-nitrofolic acidfollowing examples 1 to 4. In example 1 4-amino-2-fluoro-benzoic acidwas used instead of 4-amino-2-nitrobenzoic acid. (HPLC purity: 97.5%area, m/z=460 [M+1]⁺, ¹H-NMR (200 MHz, DMSO-d₆) [ppm]: 8.65 (s, C(7)-H,1H); 7.75 (t, N(8′)-H, 1H, exchangeable with D₂O); 7.51 (t, C(3′H), 1H);7.20 (t, N(10)H, 1H, exchangeable with D₂O); 7.03 (bs, NH₂, 2H,exchangeable with D₂O); 6.50 (m, C(5′)-H, (C(6′)-H, 2H); 4.48 (d,C(6)H₂, 2H); 4.31 (m, C(α)-H, 1H); 2.28 (m, C(β)-H₂, 2H); 1.96 (m,C(γ)-H₂, 2H).

Example 6 2′-[¹⁸F]fluoro-folic acid using 2′-nitrofolic acid

The [¹⁸F]fluoride which was trapped on an anion exchange cartridge, wasdirectly eluted into a 10 ml sealed reaction vessel using a solution ofpotassium carbonate (1 mg) and Kryptofix© 2.2.2 (5 mg) in 1.5 mlacetonitrile/water (4:1). At 85-90° C. the solvents were removed byvacuum and a stream of nitrogen. Subsequently, 1 ml of dry acetonitrilewas added three times and evaporated to dryness.

To the dry [¹⁸F]fluoride-cryptate complex the precursorN²,N,N-dimethylaminomethylene-2′-nitrofolic acid di-tert.-butylester(5.2 mg) in 0.2 ml DMF were added. The mixture is heated to 140-145° C.for 20 min.

After cooling, 8 ml water were added and the mixture was passed though areversed phase cartridge (Sep-Pak® ^(t)C18 plus, Waters AG). Thecartridge was washed three times with 8 ml of water and dried 2 min by astream of nitrogen. The ¹⁸F-labelled protected compound was eluted with2.5 ml of acetonitrile into another 10 ml sealed reaction vessel. Thevolume of acetonitrile was reduced to 0.3 ml under reduced pressure,nitrogen stream and slight warming of 80-90° C.

For hydrolysis, 0.5 ml of 4M HCl solution was added and the mixture washeated to 60° C. for 5-10 min. After cooling, the mixture is neutralizedby 0.5 ml 4M NaOH solution. 0.5 ml of 0.15M phosphate buffer solutionwas added and the mixture was filled up with HPLC solvent A to a volumeof 5 ml.

Semi-preparative HPLC purification was carried out on a RP 18 column(Phenomenex© Gemini 5μ C18, 250×10 mm) using a gradient as follows.Solvent A=0.05M phosphate buffer solution, B=methanol, 0-30 min: A:99%→40%, 30-40 min: A: 40%→10%, 40-45 min: A: 40%→99%.

The HPLC solvent of the product fraction was evaporated under reducedpressure and a stream of nitrogen at 100° C. For formulation water and0.15M phosphate buffer solution were added to the dry product and themixture was sterile filtrated.

Example 7 In vivo and ex vivo studies using 2′-[¹⁸F]fluoro-folic acid

2′-[¹⁸F]fluoro-folic acid was applied in ex vivo biodistribution studiesusing eight nude mice bearing KB xenografts tumors. ˜2 MBq of theradiotracer were injected into each animal. In a blockade group 200 μgnatural folic acid was injected 10 min prior to the radiotracer. Theanimals were scarified 90 min post injection. The folatereceptor-positive KB tumors show a high specific uptake of theradiotracer with a ratio of 86.6% specific blockade. Furthermore a highspecific uptake of 95.5% specific blockade was also found in thekidneys, which are known to express the folate receptor.

FIG. 1 shows the high specific uptake of the 2′-[¹⁸F]fluorofolic acid infolate receptor-positive tissues.

In vivo PET imaging using the 2′-[¹⁸F]fluoro-folic acid was performed innude mice bearing KB xenografts tumors. Ca. 10 MBq of the radiotracerwere injected into each animal. In the blockade group 200 μg naturalfolic acid was injected 10 min prior to the radiotracer. The PET scanswere acquired from 30 min to 90 min post injection.

PET studies using 2′-[¹⁸F]fluoro-folic acid provided excellent images ofthe KB tumors. Furthermore, the uptake is highly specific and blocked bynatural folic acid. A high specific uptake of the radiotracer was alsofound in the kidney cortex, while no uptake was found in the kidneymedulla. This pattern is consistent with the distribution of the folatereceptor and points out the high specificity of 2′-[¹⁸F]fluoro-folicacid.

FIG. 2 show PET images using 2′-[¹⁸F]fluoro-folic acid, the arrowsindicate the position of the KB xenografts tumors.

FIG. 3 shows PET images using 2′-[¹⁸F]fluoro-folic acid, the arrowsindicate the kidneys.

FIG. 4 shows ex vivo PET images of KB xenografts tumors using2′-[¹⁸F]fluoro-folic acid.

1-35. (canceled)
 36. A method for diagnostic imaging of a cell orpopulation of cells expressing a folate-receptor, said method comprising(a) administering at least one compound of formula I in an effectiveamount to achieve diagnostic imaging,

wherein A is an amino acid, X₁ to X₅ are each N, Z is anelectron-withdrawing group, R₁, R₂ are independently of each other H,Hal, O, —OR″, —NHR″, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl,C2-C12 alkenyl, C2-C12 alkynyl, (C1-C12 alkoxy)carbonyl, and (C1-C12alkylamino)carbonyl R″ is H or C1-C6 alkyl, R₃, R₄ are independently ofeach other H, formyl, iminomethyl, nitroso, C1-C12 alkyl, C1-C12 alkoxy,C1-C12 alkanoyl, halosubstituted C1-C12 alkanoyl, m is 0, 1, 2 or 3, nis 1, p is 0, 1 or 2, q has a value of 1 to 7, and r is 0 or 1, and (b)obtaining a diagnostic image of said cell or population of cells.
 37. Amethod according to claim 36, wherein the diagnostic imaging isperformed of a cell or population of cells expressing a folate-receptorin vitro.
 38. A method for in vitro detection of a cell expressing thefolate receptor in a tissue sample comprising (a) contacting said tissuesample with a compound according to formula I in an effective amount andfor a sufficient time and condition to allow binding to occur,

wherein A is an amino acid, X₁ to X₅ are each N, Z is anelectron-withdrawing group, R₁, R₂ are independently of each other H,Hal, O, —OR″, —NHR″, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl,C2-C12 alkenyl, C2-C12 alkynyl, (C1-C12 alkoxy)carbonyl, and (C1-C12alkylamino)carbonyl R″ is H or C1-C6 alkyl, R₃, R₄ are independently ofeach other H, formyl, iminomethyl, nitroso, C1-C12 alkyl, C1-C12 alkoxy,C1-C12 alkanoyl, halosubstituted C1-C12 alkanoyl, m is 0, 1, 2 or 3, nis 1, p is 0, 1 or 2, q has a value of 1 to 7, and r is 0 or 1, and (b)detecting such binding.
 39. A method of diagnostic imaging or monitoringa subject comprising (a) administering at least one compound accordingto formula I in an effective amount to achieve diagnostic imaging,

wherein A is an amino acid, X₁ to X₅ are each N, Z is anelectron-withdrawing group, R₁, R₂ are independently of each other H,Hal, O, —OR″, —NHR″, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl,C2-C12 alkenyl, C2-C12 alkynyl, (C1-C12 alkoxy)carbonyl, and (C1-C12alkylamino)carbonyl R″ is H or C1-C6 alkyl, R₃, R₄ are independently ofeach other H, formyl, iminomethyl, nitroso, C1-C12 alkyl, C1-C12 alkoxy,C1-C12 alkanoyl, halosubstituted C1-C12 alkanoyl, m is 0, 1, 2 or 3, nis 1, p is 0, 1 or 2, q has a value of 1 to 7, and r is 0 or 1, and (b)performing diagnostic imaging using PET by detecting a signal from saidat least one compound.
 40. A method according to claim 39, which is formonitoring cancer or inflammatory and autoimmune disease therapy in asubject comprising (a) administering to a subject in need thereof atleast one compound according to formula I in an effective amount toachieve diagnostic imaging in combination with a therapeutically activecompound,

wherein A is an amino acid, X₁ to X₅ are each N, Z is anelectron-withdrawing group, R₁, R₂ are independently of each other H,Hal, O, —OR″, —NHR″, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl,C2-C12 alkenyl, C2-C12 alkynyl, (C1-C12 alkoxy)carbonyl, and (C1-C12alkylamino)carbonyl R″ is H or C1-C6 alkyl, R₃, R₄ are independently ofeach other H, formyl, iminomethyl, nitroso, C1-C12 alkyl, C1-C12 alkoxy,C1-C12 alkanoyl, halosubstituted C1-C12 alkanoyl, m is 0, 1, 2 or 3, nis 1, p is 0, 1 or 2, q has a value of 1 to 7, and r is 0 or 1, and (b)performing diagnostic imaging using PET by detecting a signal from saidat least one compound to follow the course of cancer or inflammatory andautoimmune disease therapy.
 41. A method of claim 39 further comprisingdiagnosis or therapy of cancer or inflammatory and autoimmune disease bya compound other than the compound of formula I.
 42. A method accordingto claim 36, wherein the diagnostic imaging is performed of a cell orpopulation of cells expressing a folate-receptor in vivo.
 43. A methodaccording to claim 36, wherein the aminobenzoyl moiety of the compoundof formula I is substituted with fluorine-18 in the 2′-position or the6′-position.
 44. A method according to claim 36, wherein theaminobenzoyl moiety of the compound of formula I is substituted with atleast one electron-withdrawing group Z in the 2′-position or the6′-position.
 45. A method according to claim 44, wherein theelectron-withdrawing group Z is selected from the group consisting of—NO₂, —CN, —N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodoniumsalts —I⁺(R′)₂, dialkyl/-aryl silanes —SiOH(R′)₂, and silanols—SiH(R′)₂, wherein R′ is independently a straight-chain or branchedC₍₁₋₁₂₎ alkyl group or an optionally substituted carbocyclic andheterocyclic group comprising five-, six- or ten-membered ring systems.46. A method according to claim 36, wherein A is an amino acid selectedfrom glutamic acid, aspartic acid, glutamine, aspartine, lysine,arginine, cystein, and homopolymers thereof.
 47. A method according toclaim 36, wherein the compound of formula I has formula II,

wherein X₁ to X₅ are each N, X₆, X₇ are independently of each other C, Nor O, Z is an electron-withdrawing group selected from —NO₂, —CN,—N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts —I⁺(R′)₂,dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂, R′ isindependently a straight-chain or branched C₍₁₋₁₂₎ alkyl group or anoptionally substituted carbocyclic and heterocyclic group comprisingfive-, six- or ten-membered ring systems, R₁, R₂ are independently ofeach other H, Hal, O, —OR″, —NHR″, C1-C12 alkyl, C1-C12 alkoxy, C1-C12alkanoyl, C2-C12 alkenyl, C2-C12 alkynyl, (C1-C12 alkoxy)carbonyl, and(C1-C12 alkylamino)carbonyl, R″ is H or C1-C6 alkyl, R₃, R₄ areindependently of each other H, formyl, iminomethyl, nitroso, C1-C12alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, halosubstituted C1-C12 alkanoyl,R₅, R₆ are independently of each other H or straight chain or branchedC₁-C₁₂ alkyl, which is unsubstituted or substituted by at least one CN,Hal, or NO₂, and wherein one or more of embedded, non-adjacent CH2groups may independently be replaced by —O—, —CO—, —CO—O—, —CO—NR′—,—CH═CH—, —C≡C—, m is 0, 1, 2 or 3, n is 1, r is 0 or 1, p is 0, 1 or 2,and q has a value of 1 to
 7. 48. A method according to claim 47, whereinthe fluorine-18 is at the 2′- or 6′-position.
 49. A method according toclaim 36, wherein the compound of formula I has formulae III or IV,

wherein X₁ to X₅ are each N, X₆, X₇ are independently of each other C, Nor O, Z is an electron-withdrawing group selected from —NO₂, —CN,—N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts —I⁺(R′)₂,dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂, R′ isindependently a straight-chain or branched C₍₁₋₁₂₎ alkyl group or anoptionally substituted carbocyclic and heterocyclic group comprisingfive-, six- or ten-membered ring systems, R₁, R₂ are independently ofeach other H, Hal, O, —OR″, —NHR″, C1-C12 alkyl, C1-C12 alkoxy, C1-C12alkanoyl, C2-C12 alkenyl, C2-C12 alkynyl, (C1-C12 alkoxy)carbonyl, and(C1-C12 alkylamino)carbonyl, R″ is H or C1-C6 alkyl, R₃, R₄ areindependently of each other H, formyl, iminomethyl, nitroso, C1-C12alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, halosubstituted C1-C12 alkanoyl,R₅, R₆ are independently of each other H or straight chain or branchedC₁-C₁₂ alkyl, which is unsubstituted or substituted by at least one CN,Hal, or NO₂, and wherein one or more of embedded, non-adjacent CH2groups may independently be replaced by —O—, —CO—, —CO—O—, —CO—NR′—,—CH═CH—, m is 0, 1, 2, or 3, r is 0 or 1, p is 0, 1 or 2, and q has avalue of 1 to
 7. 50. A method according to claim 36, wherein thecompound of formula I has formulae V or VI,

wherein X₁ to X₅ are each N, X₆, X₇ are independently of each other C, Nor O, R₁, R₂ are independently of each other H, Hal, O, —OR″, —NHR″,C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C2-C12 alkenyl, C2-C12alkynyl, (C1-C12 alkoxy)carbonyl, and (C1-C12 alkylamino)carbonyl, R″ isH or C1-C6 alkyl, R₃, R₄ are independently of each other H, formyl,iminomethyl, nitroso, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl,halosubstituted C1-C12 alkanoyl, R₅, R₆ are independently of each otherH or straight chain or branched C₁-C₁₂ alkyl, which is unsubstituted orsubstituted by at least one CN, Hal, or NO₂, and wherein one or more ofembedded, non-adjacent CH2 groups may independently be replaced by —O—,—CO—, —CO—O—, —CO—NR′—, —CH═CH—, —C≡C—, r is 0 or 1, p is 0, 1 or 2, andq has a value of 1 to
 7. 51. A method according to claim 36, wherein thecompound of formula I has formulae VII, VIII, IX, X or XI,

wherein X₁ to X₅ are each N, X₆, X₇ are independently of each other C, Nor O, Z is an electron-withdrawing group selected from —NO₂, —CN,—N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts —I⁺(R′)₂,dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂, R′ isindependently a straight-chain or branched C₍₁₋₁₂₎ alkyl group or anoptionally substituted carbocyclic and heterocyclic group comprisingfive-, six- or ten-membered ring systems, R₁, R₂ are independently ofeach other H, Hal, O, —OR″, —NHR″, C1-C12 alkyl, C1-C12 alkoxy, C1-C12alkanoyl, C2-C12 alkenyl, C2-C12 alkynyl, (C1-C12 alkoxy)carbonyl, and(C1-C12 alkylamino)carbonyl, R″ is H or C1-C6 alkyl, R₃, R₄ areindependently of each other H, formyl, iminomethyl, nitroso, C1-C12alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, halosubstituted C1-C12 alkanoyl,R₅, R₆ are independently of each other H or straight chain or branchedC₁-C₁₂ alkyl, which is unsubstituted or substituted by at least one CN,Hal, or NO₂, and wherein one or more of embedded, non-adjacent CH2groups may independently be replaced by —O—, —CO—, —CO—O—, —CO—NR′—,—CH═CH—, n is 1, r is 0 or 1, p is 0, 1 or 2, and q has a value of 1 to7.
 52. A method according to claim 36, wherein the compound of formula Ihas formula XV,

wherein Z is an electron-withdrawing group selected from —NO₂, —CN,—N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts —I⁺(R′)₂,dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂, R′ isindependently a straight-chain or branched C₍₁₋₁₂₎ alkyl group or anoptionally substituted carbocyclic and heterocyclic group comprisingfive-, six- or ten-membered ring systems, R₅, R₆ are independently ofeach other H or straight chain or branched C₁-C₁₂ alkyl, which isunsubstituted or substituted by at least one CN, Hal, or NO₂, andwherein one or more of embedded, non-adjacent CH2 groups mayindependently be replaced by —O—, —CO—, —CO—O—, —CO—NR′—, —CH═CH—, Y₁,Y₂ are independently of each other selected from H or straight chain orbranched C₁-C₆ alkyl, R₄ is selected from H, nitroso, C₁-C₁₂ alkyl,C₁-C₁₂ alkoxy, C₁-C₁₂ alkanoyl, halosubstituted C₁-C₁₂ alkanoyl, m is 0,1, 2 or 3, and n is
 1. 53. A method according to claim 36, wherein thecompound of formula I has formula XVI,

wherein Z is an electron-withdrawing group selected from —NO₂, —CN,—N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts —I⁺(R′)₂,dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂, R′ isindependently a straight-chain or branched C₍₁₋₁₂₎ alkyl group or anoptionally substituted carbocyclic and heterocyclic group comprisingfive-, six- or ten-membered ring systems, R₃, R₄ are independently ofeach other H, formyl, iminomethyl, nitroso, C1-C12 alkyl, C1-C12 alkoxy,C1-C12 alkanoyl, halosubstituted C1-C12 alkanoyl, R₅, R₆ areindependently of each other H or straight chain or branched C₁-C₁₂alkyl, which is unsubstituted or substituted by at least one CN, Hal, orNO₂, and wherein one or more of embedded, non-adjacent CH2 groups mayindependently be replaced by —O—, —CO—, —CO—O—, —CO—NR′—, —CH═CH—, Y₁,Y₂ are independently of each other selected from H or straight chain orbranched C₁-C₆ alkyl, m is 0, 1, 2 or 3, and n is
 1. 54. A methodaccording to claim 36, wherein the compound of formula I has formulaXVII,

wherein Z is an electron-withdrawing group selected from —NO₂, —CN,—N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodonium salts —I⁺(R′)₂,dialkyl/-aryl silanes —SiOH(R′)₂, and silanols —SiH(R′)₂, R′ isindependently a straight-chain or branched C₍₁₋₁₂₎ alkyl group or anoptionally substituted carbocyclic and heterocyclic group comprisingfive-, six- or ten-membered ring systems R₅, R₆ are independently ofeach other H or straight chain or branched C₁-C₁₂ alkyl, which isunsubstituted or substituted by at least one CN, Hal, or NO₂, andwherein one or more of embedded, non-adjacent CH2 groups mayindependently be replaced by —O—, —CO—, —CO—O—, —CO—NR′—, —CH═CH—, m is0, 1, 2 or 3, and n is
 1. 55. A method according to claim 36, whereinthe compound of formula I has formula XVIII,

wherein, Z is an electron-withdrawing group preferably selected from—NO₂, —CN, —N⁺(CH₃)₃, —SO₃R′, —COOR′, —COR′, —Cl, —Br, —F, iodoniumsalts —I⁺(R′)₂, dialkyl/-aryl silanes —SiOH(R′)₂, and silanols—SiH(R′)₂, R′ is independently a straight-chain or branched C₍₁₋₁₂₎alkyl group or an optionally substituted carbocyclic and heterocyclicgroup comprising five-, six- or ten-membered ring systems, R₃ is H,methyl- or formyl-, R₅, R₆ are independently of each other H or straightchain or branched C₁-C₁₂ alkyl, which is unsubstituted or substituted byat least one CN, Hal, or NO₂, and wherein one or more of embedded,non-adjacent CH2 groups may independently be replaced by —O—, —CO—,—CO—O—, —CO—NR′—, —CH═CH—, m is 0, 1, 2 or 3, and n is 1.